Isolation of nucleic acids using liquid-liquid phase separation of pH-sensitive elastin-like polypeptides.

Extraction of nucleic acids (NAs) is critical for many methods in molecular biology and bioanalytical chemistry. NA extraction has been extensively studied and optimized for a wide range of applications and its importance to society has significantly increased. The COVID-19 pandemic highlighted the importance of early and efficient NA testing, for which NA extraction is a critical analytical step prior to the detection by methods like polymerase chain reaction. This study explores simple, new approaches to extraction using engineered smart nanomaterials, namely NA-binding, intrinsically disordered proteins (IDPs), that undergo triggered liquid-liquid phase separation (LLPS). Two types of NA-binding IDPs are studied, both based on genetically engineered elastin-like polypeptides (ELPs), model IDPs that exhibit a lower critical solution temperature in water and can be designed to exhibit LLPS at desired temperatures in a variety of biological solutions. We show that ELP fusion proteins with natural NA-binding domains can be used to extract DNA and RNA from physiologically relevant solutions. We further show that LLPS of pH responsive ELPs that incorporate histidine in their sequences can be used for both binding, extraction and release of NAs from biological solutions, and can be used to detect SARS-CoV-2 RNA in samples from COVID-positive patients.

The Sixth Sense: Self-nucleic acid sensing in the brain.

Our innate immune system uses pattern recognition receptors (PRRs) as a first line of defense to detect microbial ligands and initiate an immune response. Viral nucleic acids are key ligands for the activation of many PRRs and the induction of downstream inflammatory and antiviral effects. Initially it was thought that endogenous (self) nucleic acids rarely activated these PRRs, however emerging evidence indicates that endogenous nucleic acids are able to activate host PRRs in homeostasis and disease. In fact, many regulatory mechanisms are in place to finely control and regulate sensing of self-nucleic acids by PRRs. Sensing of self-nucleic acids is particularly important in the brain, as perturbations to nucleic acid sensing commonly leads to neuropathology. This review will highlight the role of nucleic acid sensors in the brain, both in disease and homeostasis. We also indicate the source of endogenous stimulatory nucleic acids where known and summarize future directions for the study of this growing field.

A non-lipid nucleic acid delivery vector with dendritic cell tropism and stimulation.

Rationale: Nucleic acid constructs are commonly used for vaccination, immune stimulation, and gene therapy, but their use in cancer still remains limited. One of the reasons is that systemic delivery to tumor-associated antigen-presenting cells (dendritic cells and macrophages) is often inefficient, while off-target nucleic acid-sensing immune pathways can stimulate systemic immune responses. Conversely, certain carbohydrate nanoparticles with small molecule payloads have been shown to target these cells efficiently in the tumor microenvironment. Yet, nucleic acid incorporation into such carbohydrate-based nanoparticles has proven challenging. Methods: We developed a novel approach using cross-linked bis succinyl-cyclodextrin (b-s-CD) nanoparticles to efficiently deliver nucleic acids and small-molecule immune enhancer to phagocytic cells in tumor environments and lymph nodes. Our study involved incorporating these components into the nanoparticles and assessing their efficacy in activating antigen-presenting cells. Results: The multi-modality immune stimulators effectively activated antigen-presenting cells and promoted anti-tumor immunity in vivo. This was evidenced by enhanced delivery to phagocytic cells and subsequent immune response activation in tumor environments and lymph nodes. Conclusion: Here, we describe a new approach to incorporating both nucleic acids and small-molecule immune enhancers into cross-linked bis succinyl-cyclodextrin (b-s-CD) nanoparticles for efficient delivery to phagocytic cells in tumor environments and lymph nodes in vivo. These multi-modality immune stimulators can activate antigen-presenting cells and foster anti-tumor immunity. We argue that this strategy can potentially be used to enhance anti-tumor efficacy.

[Research advances in nucleic acid drugs developed based on circular RNAs].

The development and clinical application of nucleic acid drugs has been a trendy field. One of the notable examples is mRNA vaccines, which have been used in the fighting against SARS-CoV-2. With short development cycles and mature preparation processes, mRNA vaccines demonstrate advantages in the global supply and in response to virus mutations. Circular RNAs (circRNAs) are a group of nucleic acid molecules with more stable structure, longer half-life, and weaker immunogenicity than mRNAs. Studies have proven that circRNAs can efficiently express protein products, indicating their potential in drug development. Despite extensive studies on the biogenesis and biological functions of circRNAs, there is limited research on developing nucleic acid drugs based on circRNAs. This article provides an overview of circRNAs, including their basic information, synthesis routes, and mechanisms, and discusses the future development directions of this field, hoping to provide inspiration for the research and development of drugs based on circRNAs.

Nucleic Acid-Binding Dyes as Versatile Photocatalysts for Atom-Transfer Radical Polymerization.

Nucleic acid-binding dyes (NuABDs) are fluorogenic probes that light up after binding to nucleic acids. Taking advantage of their fluorogenicity, NuABDs have been widely utilized in the fields of nanotechnology and biotechnology for diagnostic and analytical applications. We demonstrate the potential of NuABDs together with an appropriate nucleic acid scaffold as an intriguing photocatalyst for precisely controlled atom-transfer radical polymerization (ATRP). Additionally, we systematically investigated the thermodynamic and electrochemical properties of the dyes, providing insights into the mechanism that drives the photopolymerization. The versatility of the NuABD-based platform was also demonstrated through successful polymerizations using several NuABDs in conjunction with diverse nucleic acid scaffolds, such as G-quadruplex DNA or DNA nanoflowers. This study not only extends the horizons of controlled photopolymerization but also broadens opportunities for nucleic acid-based materials and technologies, including nucleic acid-polymer biohybrids and stimuli-responsive ATRP platforms.

Double synergic chitosan-coated poly (lactic-co-glycolic) acid nanospheres loaded with nucleic acids as an intranasally administered vaccine delivery system to control the infection of foot-and-mouth disease virus.

BACKGROUND & AIMS: The spread of foot-and-mouth disease virus (FMDV) through aerosol droplets among cloven-hoofed ungulates in close contact is a major obstacle for successful animal husbandry. Therefore, the development of suitable mucosal vaccines, especially nasal vaccines, to block the virus at the initial site of infection is crucial. PATIENTS AND METHODS: Here, we constructed eukaryotic expression plasmids containing the T and B-cell epitopes (pTB) of FMDV in tandem with the molecular mucosal adjuvant Fms-like tyrosine kinase receptor 3 ligand (Flt3 ligand, FL) (pTB-FL). Then, the constructed plasmid was electrostatically attached to mannose-modified chitosan-coated poly(lactic-co-glycolic) acid (PLGA) nanospheres (MCS-PLGA-NPs) to obtain an active nasal vaccine targeting the mannose-receptor on the surface of antigen-presenting cells (APCs). RESULTS: The MCS-PLGA-NPs loaded with pTB-FL not only induced a local mucosal immune response, but also induced a systemic immune response in mice. More importantly, the nasal vaccine afforded an 80% protection rate against a highly virulent FMDV strain (AF72) when it was subcutaneously injected into the soles of the feet of guinea pigs. CONCLUSIONS: The nasal vaccine prepared in this study can effectively induce a cross-protective immune response against the challenge with FMDV of same serotype in animals and is promising as a potential FMDV vaccine.

Chemistry and Biology of Noncanonical Nucleic Acid Structures: From Physicochemical Properties to Therapeutic Applications.

The aim of this Special Issue is to highlight significant and new aspects concerning the chemistry and biology of noncanonical nucleic acid structures, with emphasis on their structure, stability, and conformational equilibria, as well as on the biological relevance of their interactions with proteins and ligands [...].

Nucleic acid liquids.

The confluence of recent discoveries of the roles of biomolecular liquids in living systems and modern abilities to precisely synthesize and modify nucleic acids (NAs) has led to a surge of interest in liquid phases of NAs. These phases can be formed primarily from NAs, as driven by base-pairing interactions, or from the electrostatic combination (coacervation) of negatively charged NAs and positively charged molecules. Generally, the use of sequence-engineered NAs provides the means to tune microsopic particle properties, and thus imbue specific, customizable behaviors into the resulting liquids. In this way, researchers have used NA liquids to tackle fundamental problems in the physics of finite valence soft materials, and to create liquids with novel structured and/or multi-functional properties. Here, we review this growing field, discussing the theoretical background of NA liquid phase separation, quantitative understanding of liquid material properties, and the broad and growing array of functional demonstrations in these materials. We close with a few comments discussing remaining open questions and challenges in the field.

Self-Powered Microfluidics for Point-of-Care Solutions: From Sampling to Detection of Proteins and Nucleic Acids.

Self-powered microfluidics presents a revolutionary approach to address the challenges of healthcare in decentralized and point-of-care settings where limited access to resources and infrastructure prevails or rapid clinical decision-making is critical. These microfluidic systems exploit physical and chemical phenomena, such as capillary forces and surface tension, to manipulate tiny volumes of fluids without the need for external power sources, making them cost-effective and highly portable. Recent technological advancements have demonstrated the ability to preprogram complex multistep liquid operations within the microfluidic circuit of these standalone systems, which enabled the integration of sensitive detection and readout principles. This chapter first addresses how the accessibility to in vitro diagnostics can be improved by shifting toward decentralized approaches like remote microsampling and point-of-care testing. Next, the crucial role of self-powered microfluidic technologies to enable this patient-centric healthcare transition is emphasized using various state-of-the-art examples, with a primary focus on applications related to biofluid collection and the detection of either proteins or nucleic acids. This chapter concludes with a summary of the main findings and our vision of the future perspectives in the field of self-powered microfluidic technologies and their use for in vitro diagnostics applications.

Nucleic acid-based electrochemical biosensors.

Colorectal cancer, breast cancer, oxidative DNA damage, and viral infections are all significant and major health threats to human health, presenting substantial challenges in early diagnosis. In this regard, a wide range of nucleic acid-based electrochemical platforms have been widely employed as point-of-care diagnostics in health care and biosensing technologies. This review focuses on biosensor design strategies, underlying principles involved in the development of advanced electrochemical genosensing devices, approaches for immobilizing DNA on electrode surfaces, as well as their utility in early disease diagnosis, with a particular emphasis on cancer, leukaemia, oxidative DNA damage, and viral pathogen detection. Notably, the role of biorecognition elements and nanointerfaces employed in the design and development of advanced electrochemical genosensors for recognizing biomarkers related to colorectal cancer, breast cancer, leukaemia, oxidative DNA damage, and viral pathogens has been extensively reviewed. Finally, challenges associated with the fabrication of nucleic acid-based biosensors to achieve high sensitivity, selectivity, a wide detection range, and a low detection limit have been addressed. We believe that this review will provide valuable information for scientists and bioengineers interested in gaining a deeper understanding of the fabrication and functionality of nucleic acid-based electrochemical biosensors for biomedical diagnostic applications.

Development of Aptamer-DNAzyme based metal-nucleic acid frameworks for gastric cancer therapy.

The metal-nucleic acid nanocomposites, first termed metal-nucleic acid frameworks (MNFs) in this work, show extraordinary potential as functional nanomaterials. However, thus far, realized MNFs face limitations including harsh synthesis conditions, instability, and non-targeting. Herein, we discover that longer oligonucleotides can enhance the synthesis efficiency and stability of MNFs by increasing oligonucleotide folding and entanglement probabilities during the reaction. Besides, longer oligonucleotides provide upgraded metal ions binding conditions, facilitating MNFs to load macromolecular protein drugs at room temperature. Furthermore, longer oligonucleotides facilitate functional expansion of nucleotide sequences, enabling disease-targeted MNFs. As a proof-of-concept, we build an interferon regulatory factor-1(IRF-1) loaded Ca2+/(aptamer-deoxyribozyme) MNF to target regulate glucose transporter (GLUT-1) expression in human epidermal growth factor receptor-2 (HER-2) positive gastric cancer cells. This MNF nanodevice disrupts GSH/ROS homeostasis, suppresses DNA repair, and augments ROS-mediated DNA damage therapy, with tumor inhibition rate up to 90%. Our work signifies a significant advancement towards an era of universal MNF application.

RmsdXNA: RMSD prediction of nucleic acid-ligand docking poses using machine-learning method.

Small molecule drugs can be used to target nucleic acids (NA) to regulate biological processes. Computational modeling methods, such as molecular docking or scoring functions, are commonly employed to facilitate drug design. However, the accuracy of the scoring function in predicting the closest-to-native docking pose is often suboptimal. To overcome this problem, a machine learning model, RmsdXNA, was developed to predict the root-mean-square-deviation (RMSD) of ligand docking poses in NA complexes. The versatility of RmsdXNA has been demonstrated by its successful application to various complexes involving different types of NA receptors and ligands, including metal complexes and short peptides. The predicted RMSD by RmsdXNA was strongly correlated with the actual RMSD of the docked poses. RmsdXNA also outperformed the rDock scoring function in ranking and identifying closest-to-native docking poses across different structural groups and on the testing dataset. Using experimental validated results conducted on polyadenylated nuclear element for nuclear expression triplex, RmsdXNA demonstrated better screening power for the RNA-small molecule complex compared to rDock. Molecular dynamics simulations were subsequently employed to validate the binding of top-scoring ligand candidates selected by RmsdXNA and rDock on MALAT1. The results showed that RmsdXNA has a higher success rate in identifying promising ligands that can bind well to the receptor. The development of an accurate docking score for a NA-ligand complex can aid in drug discovery and development advancements. The code to use RmsdXNA is available at the GitHub repository https://github.com/laiheng001/RmsdXNA.

Catalytic Hairpin Assembly-Based Self-Ratiometric Gel Electrophoresis Detection Platform for Reliable Nucleic Acid Analysis.

The development of gel electrophoresis-based biodetection assays for point-of-care analysis are highly demanding. In this work, we proposed a ratiometric gel electrophoresis-based biosensing platform by employing catalytic hairpin assembly (CHA) process functions as both the signal output and the signal amplification module. Two types of nucleic acids, DNA and miRNA, are chosen for demonstration. The proposed strategy indeed provides a new paradigm for the design of a portable detection platform and may hold great potential for sensitive diagnoses.

Sensitive, precise fingerprint profiling for monosaccharide analysis of Bacillus Calmette-Guérin polysaccharide and nucleic acid isolates.

A sensitive and precise HPLC-DAD method with pre-column PMP derivatization was established and validated, for analyzing the polysaccharides in Bacillus Calmette-Guérin polysaccharide and nucleic acid (BCG-PSN) isolates, after acid hydrolysis. And the HPLC fingerprint profiling was used to analyze its monosaccharide composition. The monosaccharide concentration-peak area calibration curve was of good linearity (R2 > 0.99), over the range of 0.016-0.08 mg/mL for mannose or 0.24-1.20 mg/mL for glucose, with high recovery of 93-105 % for quality control samples. The intra-day RSD values of mannose and glucose concentration were less than 2.5 % and 2.1 %, respectively, and their inter-day RSD values were less than 4.3 % and 2.2 %, respectively, and remained stable for up to 14 days. This method also remained durable against changes in chromatographic parameters, but it's susceptible to the flow rate of mobile phase. Additionally, the method was applied to analyze the content of mannose and glucose in 22 batches BCG-PSN powder and 17 batches BCG-PSN injection. The results showed that the HPLC-DAD fingerprint spectra of all the BCG-PSN powder and BCG-PSN injection samples had a high degree of similarity, with the similar indexes up to 0.999 and 0.998, respectively. The HPLC-DAD method with pre-column PMP derivatization is highly rapid, effective, visual, and accurate for determination of monosaccharide contents. The validated method was successfully applied to the analysis of polysaccharide in both BCG-PSN powder and injection.

Investigating the biochemical response of proton minibeam radiation therapy by means of synchrotron-based infrared microspectroscopy.

The biology underlying proton minibeam radiation therapy (pMBRT) is not fully understood. Here we aim to elucidate the biological effects of pMBRT using Fourier Transform Infrared Microspectroscopy (FTIRM). In vitro (CTX-TNA2 astrocytes and F98 glioma rat cell lines) and in vivo (healthy and F98-bearing Fischer rats) irradiations were conducted, with conventional proton radiotherapy and pMBRT. FTIRM measurements were performed at ALBA Synchrotron, and multivariate data analysis methods were employed to assess spectral differences between irradiation configurations and doses. For astrocytes, the spectral regions related to proteins and nucleic acids were highly affected by conventional irradiations and the high-dose regions of pMBRT, suggesting important modifications on these biomolecules. For glioma, pMBRT had a great effect on the nucleic acids and carbohydrates. In animals, conventional radiotherapy had a remarkable impact on the proteins and nucleic acids of healthy rats; analysis of tumour regions in glioma-bearing rats suggested major nucleic acid modifications due to pMBRT.

Sampling efficiency and nucleic acid stability during long-term sampling with different bioaerosol samplers.

Aerosol microbiome studies have received increased attention as technological advancements have made it possible to dive deeper into the microbial diversity. To enhance biomass collection for metagenomic sequencing, long-term sampling is a common strategy. While the impact of prolonged sampling times on microorganisms' culturability and viability is well-established, its effect on nucleic acid stability remains less understood but is essential to ensure representative sample collection. This study evaluated four air samplers (SKC BioSampler, SASS3100, Coriolis µ, BioSpot-VIVAS 300-P) against a reference sampler (isopore membrane filters) to identify nucleic acid stability during long-term sampling. Physical sampling efficiencies determined with a fluorescent tracer for three particle sizes (0.8, 1, and 3 µm), revealed high efficiencies (> 80% relative to reference) for BioSampler, SASS3100, and BioSpot-VIVAS for all particle sizes, and for Coriolis with 3 µm particles. Coriolis exhibited lower efficiency for 0.8 µm (7%) and 1 µm (50%) particles. During 2-h sampling with MS2 and Pantoea agglomerans, liquid-based collection with Coriolis and BioSampler showed a decrease in nucleic acid yields for all test conditions. BioSpot-VIVAS displayed reduced sampling efficiency for P. agglomerans compared to MS2 and the other air samplers, while filter-based collection with SASS3100 and isopore membrane filters, showed indications of DNA degradation for 1 µm particles of P. agglomerans after long-term sampling. These findings show that long-term air sampling affects nucleic acid stability in both liquid- and filter-based collection methods. These results highlight bias produced by bioaerosol collection and should be considered when selecting an air sampler and interpreting aerosol microbiome data.

Metagenomics analysis reveals effects of salinity fluctuation on diversity and ecological functions of high and low nucleic acid content bacteria.

Salinity is a critical environmental factor in marine ecosystems and has complex and wide-ranging biological effects. However, the effects of changing salinity on diversity and ecological functions of high nucleic acid (HNA) and low nucleic acid (LNA) bacteria are not well understood. In this study, we used 16S rRNA sequencing and metagenomic sequencing analysis to reveal the response of HNA and LNA bacterial communities and their ecological functions to salinity, which was decreased from 26 ‰ to 16 ‰. The results showed that salinity changes had significant effects on the community composition of HNA and LNA bacteria. Among LNA bacteria, 14 classes showed a significant correlation between relative abundance and salinity. Salinity changes can lead to the transfer of some bacteria from HNA bacteria to LNA bacteria. In the network topology relationship, the complexity of the network between HNA and LNA bacterial communities gradually decreased with decreased salinity. The abundance of some carbon and nitrogen cycling genes in HNA and LNA bacteria varied with salinity. Overall, this study demonstrates the effects of salinity on diversity and ecological functions and suggests the importance of salinity in regulating HNA and LNA bacterial communities and functions.

A comprehensive review of protein-centric predictors for biomolecular interactions: from proteins to nucleic acids and beyond.

Proteins interact with diverse ligands to perform a large number of biological functions, such as gene expression and signal transduction. Accurate identification of these protein-ligand interactions is crucial to the understanding of molecular mechanisms and the development of new drugs. However, traditional biological experiments are time-consuming and expensive. With the development of high-throughput technologies, an increasing amount of protein data is available. In the past decades, many computational methods have been developed to predict protein-ligand interactions. Here, we review a comprehensive set of over 160 protein-ligand interaction predictors, which cover protein-protein, protein-nucleic acid, protein-peptide and protein-other ligands (nucleotide, heme, ion) interactions. We have carried out a comprehensive analysis of the above four types of predictors from several significant perspectives, including their inputs, feature profiles, models, availability, etc. The current methods primarily rely on protein sequences, especially utilizing evolutionary information. The significant improvement in predictions is attributed to deep learning methods. Additionally, sequence-based pretrained models and structure-based approaches are emerging as new trends.

Tetrahedral framework nucleic acids/hyaluronic acid-methacrylic anhydride hybrid hydrogel with antimicrobial and anti-inflammatory properties for infected wound healing.

Bacterial resistance and excessive inflammation are common issues that hinder wound healing. Antimicrobial peptides (AMPs) offer a promising and versatile antibacterial option compared to traditional antibiotics, with additional anti-inflammatory properties. However, the applications of AMPs are limited by their antimicrobial effects and stability against bacterial degradation. TFNAs are regarded as a promising drug delivery platform that could enhance the antibacterial properties and stability of nanodrugs. Therefore, in this study, a composite hydrogel (HAMA/t-GL13K) was prepared via the photocross-linking method, in which tFNAs carry GL13K. The hydrogel was injectable, biocompatible, and could be instantly photocured. It exhibited broad-spectrum antibacterial and anti-inflammatory properties by inhibiting the expression of inflammatory factors and scavenging ROS. Thereby, the hydrogel inhibited bacterial infection, shortened the wound healing time of skin defects in infected skin full-thickness defect wound models and reduced scarring. The constructed HAMA/tFNA-AMPs hydrogels exhibit the potential for clinical use in treating microbial infections and promoting wound healing.